HBsAg无症状携带者乙型肝炎病毒核心基因启动子突变的研究

目的了解HBsAg无症状携带者乙型肝炎病毒(HBV)核心基因启动子双突变(nt1762 A→T,nt1764 G→A)发生情况及其与基因型、病毒量和HBeAg的关系。方法 HBsAg无症状携带者从隆安研究队列中选择,用套式聚合酶链反应(nested PCR)对研究对象血清HBV核心基因启动子、S基因扩增、序列分析及基因分型,用HBV引物和双标记的TaqMan探针扩增和定量病毒DNA。结果观察开始时核心基因启动子为野毒株的109例观察对象,3年后双突变平均年发生率为2.8%;观察开始时已发生nt1762或nt1764点突变的59例对象,3年后另一个位点也发生突变的平均年发生率为6.8%,两率差异有统计学意义(χ2=5.109 0,P<0.05)。nt1762 A→T突变组HBeAg阳性率(45.5%,10/22)与nt1764 G→A突变组HBeAg阳性率(10.8%,4/37)差异有统计学意义(χ2=9.149,P<0.05)。20例基因型B未发生双突变,重组基因型年双突变率为4.8%(2/14),基因型C年突变率为3.1%(7/75),各基因型间突变率差异无统计学意义(P>0.05)。5例双突变发生后HBeAg仍然阳性者病毒量有下降趋势,而另5例突变发生后HBeAg阴性者其病毒量有升有降。结论 HBV核心基因启动子双突变年发生率较高,这两个位点中的任何一个发生突变是双突变的过渡形式,双突变与基因型无关,nt1764 G→A突变与HBeAg阴性有关。     

Increased hepatitis B virus quasispecies diversity is correlated with liver fibrosis progression

Host immune response and viral factors are involved in disease progression in patients with chronic hepatitis B virus (HBV) infection. However, the relationship between HBV quasispecies and liver fibrosis progression remains unclear. In this study, 447 patients with chronic HBV infection, including 239 with chronic hepatitis B (CHB), 104 with liver cirrhosis (LC) and 104 with hepatocellular carcinoma (HCC) were enrolled. The 239 CHB patients were divided into groups F1, F2, and F3 according to liver fibrosis score. Four fragments of the HBV genome were determined and analyzed using next-generation sequencing. Specific mutations, such as A1762T, G1764A and G1896A, in the BCP/PC region were more common in patients with advanced liver disease and formed the majority of the viral quasispecies pool in patients with LC and HCC. The viral complexity and diversity increased as the fibrosis progressed, especially in patients with CHB who were comparable in age but at different stages of fibrosis. Patients with early-stage fibrosis experienced higher purifying selection pressure in the four sequenced regions, whereas different protein-coding region experienced different negative selection with disease progression. HBV quasispecies diversity may increase fibrosis progression in CHB patients with aging under immune selection.     

15 year fulminant hepatitis B follow-up in Belgium: Viral evolution and signature of demographic change

Fulminant hepatitis among different clinical outcomes of hepatitis B virus infection is very rare and manifests high mortality rate, however it has not been investigated in Belgian inhabitants yet. In the frame of a retrospective study between 1995 and 2010, 80 serum samples (in some cases serial samples) archived in Biobank, were collected from 24 patients who had clinically developed fulminant infection of hepatitis B virus. In total, 33 hepatitis B virus (HBV) strains (31 full-length genome and 2 partial viral genes) of different HBV genotypes and subgenotypes including A2, B2, D1, D2, D3 and E, were amplified, sequenced and phylogenetically analyzed. HBV isolated strains from native and exotic patients were characterized by genome variations associated with viral invasiveness. Although several mutations at nucleotide and protein levels were detected, evolutionary analyses revealed a negative selective pressure over the viral genomes. This study revealed influence of immigration through a steady change in the viral epidemiological profile of the Belgian population.     

乙型肝炎病毒基因型和血清亚型在我国部分地区的分布及其特点

目的 研究乙型肝炎病毒（hepatitis B virus,HBV）基因型和血清亚型的分布特点及其基因的相关性。方法 湖南、广西、河南和河北4省（区）25个县（市）280例慢性HBV携带者,应用聚合酶链反应（PCR）扩增和脱氧核糖核酸（DNA）序列分析,确定HBV基因型和血清亚型。结果 HBV基因型B、C和D均有分布,分别占29.3%、67.9%和2.9%,其中B和C为优势基因型。adr、adw2、ayr、aywl、ayw2和ayw3等6种血清亚型均有分布,其中adr和adw2为优势血清亚型,分别占64.3%和31.4%。基因型B与adw2血清亚型,基因型C与adr血清亚型有非常密切的基因相关性。280例HBV携带者表面抗原基因序列每100个核苷酸的平均置换频数为2.94。基因型B(adw2血清亚型）毒株核苷酸置换频数为5.63(5.48）,而基因型C（adr血清亚型）仅为1.6(1.51）。结论 HBV基因型和血清亚型的分布均有明显的地区性;基因型B及相对应的adw2血清亚型毒株的基因可变性明显高于基因型C及相应的adr血清亚型。     

一起氟乙酰胺蓄积性中毒分析

氟乙酰胺急性中毒时有发生 ,其中毒者临床表现与实验室诊断技术已为预防医学工作者所掌握 ,但氟乙酰胺蓄积性中毒十分罕见 ,经检索国内尚无个案报道。现就集宁市发生的一起氟乙酰胺蓄积性中毒分析报告如下。患者男性 6 3岁 ,2 0 0 1年 6月 2 0日由家属陪送入院 ,主诉神志有时不清 ,妄语 ,肢体阵发性抽搐 ,大小便失禁 ,视力模糊 ,病史已有 4个多月 ,院方即按颠痫收住入院 ,经过 1周对症治疗但疗效甚微 ,遂建议其转入精神病院 ,经精神病专家会诊 ,发现患者有接触鼠药史 ,于是要求卫生防疫部门协助检验。该病例系鼠药商贩 ,同时经销数种鼠药 ,…     

Impact of HBV genotypes A and D genetic variability on infection evolution

HBV is characterized by a high genetic variability, which is the basis of its classification into eight genotypes (A–H). HBV infection is associated with different outcomes, from self-limiting acute hepatitis to active chronic hepatitis, asymptomatic carriage, and occult infection.The aim of this study was to analyze the genetic variability of HBV genotypes A and D isolates from 79 cases of self-limiting acute hepatitis and chronic hepatitis, in order to identify HBV variants associated with resolution or chronicity of acute HBV infection. The entire preS–S sequence and a fragment of 346 bp of the preC–C region, containing Enhancer II and Basal Core Promoter sequences, were analyzed. A phylogenetic analysis of preS/S region showed that the 45.45% (15/33) of isolates from acute hepatitis cases were genotype A compared to 8.69% (4/46) of chronic hepatitis cases. (p = 0.0002). Mutations associated with immune-escape (T131N, D144A/E, G145K), amino acid polymorphisms in “a determinant” domain of S protein and mutations/deletions in preC/C region were found in isolates from acute and chronic hepatitis B cases. In this study mutations/deletions in preS–S and preC–C regions, usually associated with fulminant acute hepatitis, advanced forms of liver disease and increased risk for HCC, were identified in HBV strains of genotype A and D obtained both from patients with self-limiting acute HBV infection and from persistent infected patients. This founding probably is due to the natural viral evolution under host immune response and to the circulation of a wide variety of HBV strains in our geographic area because of the ancient introduction of genotype D and the migrant fluxes from North Africa. Moreover, the analysis of circulation of new HBV antigenic variants is fundamental for the epidemiological surveys and for the evaluation of the impact of viral evolution on vaccine prophylaxis strategies.     

HBV基因型和肝细胞癌的研究现状

目前HBV可分为A～H8种基因型,基因型可分出亚型,HBV基因型及基因亚型呈现显著的地理区域性分布。感染基因型C比B的患者有更高的基因变异率、更高的HBV DNA滴度、更严重的肝损害、更低的抗病毒应答率,HBV基因型与肝细胞癌（HCC）相关。     

Evolution of cytotoxic T-lymphocyte epitopes in hepatitis B virus.

In hepatitis B virus (HBV), while mutations that escape from cytotoxic T-lymphocyte (CTL) recognition have been described it has been difficult to determine how natural selection by host CTL has influenced long-term evolution of HBV. We used statistical analysis of published HBV genomic sequences to examine the role of natural selection in evolution of CTL epitopes. Based on a phylogenetic analysis, we identified 25 pairs of closely related genomes isolated from different HBV genotypes and examined pattern of nucleotide substitution in genomic regions encoding well-characterized CTL epitopes. On average, both epitope and non-epitope regions are subject to purifying selection acting at non-synonymous sites. However, certain CTL epitopes showed a pattern of nucleotide substitution suggesting repeated positive selection across the population. The results support the hypothesis that CTL-driven selection has been an important factor in long-term evolution of HBV.     

乙型肝炎病毒e抗原前C区基因变异的血清型和基因型分布特点研究

目的分析比较不同血清型和基因型的乙型肝炎病毒(HBV)e抗原前C区基因序列,了解其特点及差异。方法在12条不同血清型和30条不同基因型的HBV全序列中,比较e抗原前C区编码基因。同时用Blast程序查找并分析与e抗原前C区变异基因相似的核苷酸序列,试图比较不同序列与慢性病毒性肝炎病情的关系。结果e抗原前C区编码基因相对保守,仅在Adw血清型出现1个6核苷酸的插入突变,在B基因型出现1个核苷酸的突变;核苷酸Nt1896位变异(T-A)可出现在血清型Adr和Adw型,和A、B、C、D、G基因型;而Nt1858位(T-C)变异见于Adw血清型,和A、F、H基因型。结论前-C区基因变异主要有插入突变和点突变两种类型,两者在不同血清型和基因型的HBV基因组中有出现频率不同,而不同变异的临床意义可能有所差别。     

慢性乙型肝炎临床表现形式与乙肝病毒基因型的关系分析

目的:探讨慢性乙型肝炎基因型与慢性乙型肝炎临床表现类型的关系。方法:对35例慢性乙型肝炎轻度,32例慢乙肝中度以及30例慢乙肝重度患者的血清采用荧光定量PCR技术进行乙肝病毒基因型的检查,分析慢性乙型肝炎的三种表现与乙肝病毒基因型的关系。结果:97例慢性乙肝患者共检出B型基因为39例(40.21%),C型基因为58例(59.79%),在慢性乙型肝炎轻度、中度、重度各组间基因型B和C分布差别无统计学意义(P>0.05),B和C基因型中HBeAg和HBV-DNA载量的表达有差别(P<0.05),C基因型的HBeAg和HBV-DNA载量表达要高于B基因型。结论:本地区慢性乙型肝炎基因型以B和C感染为主,基因型与慢性乙型肝炎轻、中、重分级无关系,C型基因的HBeAg和HBV-DNA载量表达要高于B基因型。     

单纯乙型肝炎疫苗免疫后表面抗原携带者“a”决定簇基因变异的分子流行病学特征

目的　了解单纯乙型肝炎疫苗免疫失败后表面抗原 ( HBs Ag)携带者和未免疫携带者 HBs Ag基因变异的流行特点及乙型肝炎病毒 ( HBV)基因型和血清亚型的分布特点。　方法　应用直接测序和基因序列特异固相聚合酶链反应( SS- SPPCR)方法 ,分别检测 69例单纯乙型肝炎疫苗免疫后携带者和 83例未免疫携带者 ,乙型肝炎病毒 HBs Ag“a”决定簇氨基酸的变异及 HBV基因型和血清亚型的分布。　结果　 HBV基因型 B、C和 D均存在 ,其中 B和 C为优势基因型 ;血清亚型 adr、adw2、avr、ayw1、ayw2和 ayw3均存在 ,其中 adr和 adw2为优势血清亚型。直接测序检测的乙型肝炎疫苗免疫后携带者 HBs Ag“a”决定簇氨基酸变异率明显高于未免疫携带者对照 ,变异率分别为 3 1.9% ( 2 2 / 69)和 8.4 % ( 7/ 83 )。14 5、12 6和 13 3位是最常见的氨基酸变异点。应用更为敏感的 SS- SPPCR法分别检测 14 5和 12 6位氨基酸点突变的变异率 ,各组间差别不明显。经直接测序法检测 ,免疫后携带者基因变异危险度 ( OR)是未免疫携带者的 5 .1倍。按基因型和血清亚型分层分析 ,基因型 B和 adw2血清亚型携带者受免疫后出现基因变异的危险度明显高于未免疫携带者 ,分别为3 1.7和 2 8.9。　结论　接种乙型肝炎疫苗者可引起表面抗原基因的免疫逃避     

Subgenotype D5, BCP and MHR mutations in hepatic complications among hepatitis B virus infected patients from Orissa,India

The study was undertaken to investigate the clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP), precore (PC) and surface gene mutations in HBV infected patients from Orissa, southeastern India. HBV infections were identified by serology testing and HBV DNA amplification by polymerase chain reaction among the 152 patients. After sequencing, surface gene mutation were studied by sequence analysis as well as by using BLOSUM scores and BCP mutations were studied only by sequence analysis. A high proportion of HBV/D5 (66.0%) was found among the study samples having significant relation with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients (p < 0.05). The BCP mutation, TA (81.4%) and C1753/TA (75.0%) was found in significant proportion (p < 0.05) among HCC cases and in fact a gradual increase in these mutations were noted between inactive carriers (IC) to HCC group and also showed higher viral load. An increasing trend of major hydrophilic region (MHR) mutations in S gene was also observed from IC (56.0%) to chronic liver disease (CLD) (60.4%) to LC (72.4%) to HCC (95.0%) patients. In conclusion, our study suggests that the predominant HBV subgenotype HBV/D5 with high viral load and BCP mutations (double and triple) and high mutations in MHR region was significantly associated with advanced liver disease (LC and HCC) and might act as predictor of severe hepatic complications.     

乙型肝炎病毒前C区/BCP区突变对血清HBV DNA含量的影响

目的利用基因芯片技术检测乙型肝炎病毒(HBV)前C区/BCP区基因突变,探讨前C区/BCP区基因突变后血清HBV DNA水平的变化。方法应用基因芯片技术检测95例慢性乙肝的HBV前C区1896、1814及HBV C基因启动子(BCP)1762、1764四位点突变,同时用荧光定量PCR检测血清HBV DNA含量。结果95份血清标本中,有91份分别检测到了HBV前C区/BCP区基因突变,阳性率95.8%,其中G1896A突变33例(36.3%),A1762T G1764A联合突变64例(70.3%),G1896A A1762T G1764A联合突变22例(24.2%),未检测到A1814C突变毒株。G1896A A1762T G1764A联合突变后,血清HBV DNA水平较未变异组降低(P<0.05),其它各组则无显著变化。结论应用基因芯片法可同时检测HBV多个突变位点。乙型肝炎病毒前C区/BCP区突变对血清HBV DNA含量有一定的影响。     

Hepatitis B virus (HBV) genotypes in the Czech Republic

Hepatitis B virus (HBV) was genotyped based on sequencing and analysis of nucleotide sequences of HBV S gene in sera samples from 176 patients with viral hepatitis B from all over the Czech Republic. The most frequent genotype A was detected in 118 (67.1%) of the patients, followed by genotype D found in 50 (28.4%) of the patients. Genotypes B and C were identified in 6 (3.4%) and 2 (1.1%) of the patients, respectively. The incidence of genotype A statistically significantly increases with age. There is no statistically significant correlation between genotypes A and D and gender or locality (region). Genotypes B and C, found mainly in South East Asia, were first detected in the Czech Republic. Detection of these genotypes in both foreigners and Czech population is suggestive not only of import but also of transmission of these genotypes in the Czech Republic. Fylogenetic analysis documents dissimilarity levels between HBV isolates. Based on the amino sequence derived, the percentages of HBV subtype determinants d (68.6%) and y (31.4%) were established. The gene S sequencing analysis proved suitable for HBV genotyping.     

Non-A hepatitis B virus genotypes in antenatal clinics, United Kingdom

In the United Kingdom, the National Screening Programme for identification of hepatitits B virus (HBV) infection in pregnant women uses HBV e antigen (HBeAg) and antibody to HBeAg (anti-HBe) as markers of infectivity to determine use of immunoglobulin for hepatitis B. Serum samples from 114 HBV-infected women were analyzed. Viral loads correlated with HBeAg/anti-HBe status and viral genotypes. Among 95 mothers whose serum contained anti-HBe, viral loads ranged between undetectable and 8.6 x 10(6) IU/mL (median 228 IU/mL). Ten (10.5%) of these mothers had plasma viral loads >10(4) IU/mL; 6 were infected with genotype E and one each with genotypes A, B, C, and D. All viruses had precore stop codon or basal core promoter mutations. Preponderance of genotypes other than A among antenatal mothers in the United Kingdom reflects increasing globalization and trends in immigration. HBeAg serostatus is no longer sufficiently accurate for inferring potential infectivity of pregnant HBV carriers.     

Dynamics of Hepatitis D (delta) virus genotype 3 in the Amazon region of South America

Hepatitis delta virus (HDV) is widely distributed and associated with fulminant hepatitis epidemics in areas with high prevalence of HBV. Several studies performed in the 1980s showed data on HDV infection in South America, but there are no studies on the viral dynamics of this virus. The aim of this study was to conduct an evolutionary analysis of hepatitis delta genotype 3 (HDV/3) prevalent in South America: estimate its nucleotide substitution rate, determine the time of most recent ancestor (TMRCA) and characterize the epidemic history and evolutionary dynamics. Furthermore, we characterized the presence of HBV/HDV infection in seven samples collected from patients who died due to fulminant hepatitis from Amazon region in Colombia and included them in the evolutionary analysis. This is the first study reporting HBV and HDV sequences from the Amazon region of Colombia. Of the seven Colombian patients, five were positive for HBV-DNA and HDV-RNA. Of them, two samples were successfully sequenced for HBV (subgenotypes F3 and F1b) and the five samples HDV positive were classified as HDV/3. By using all HDV/3 available reference sequences with sampling dates (n = 36), we estimated the HDV/3 substitution rate in 1.07 × 10⁻³ substitutions per site per year (s/s/y), which resulted in a time to the most recent common ancestor (TMRCA) of 85 years. Also, it was determined that HDV/3 spread exponentially from early 1950s to the 1970s in South America. This work discusses for the first time the viral dynamics for the HDV/3 circulating in South America. We suggest that the measures implemented to control HBV transmission resulted in the control of HDV/3 spreading in South America, especially after the important raise in this infection associated with a huge mortality during the 1950s up to the 1970s. The differences found among HDV/3 and the other HDV genotypes concerning its diversity raises the hypothesis of a different origin and/or a different transmission route.     

长沙地区无偿献血人群隐匿性乙型肝炎病毒感染情况分析

目的 了解长沙地区无偿献血人群隐匿性乙型肝炎病毒感染(occult hepatitis B virus infection,OBI)流行情况,探讨HBV基因型分布特征和S区氨基酸突变的情况。方法 对长沙地区检测结果为HBsAg-/HBV DNA+的无偿献血血液样本进行HBV血清标志物检测,对其中的OBI样本进行HBV病毒载量检测和S区基因扩增,分析血清学标志物抗HBs与病毒载量检出与否的关系,并对扩增产物进行HBV基因分型和突变位点分析。结果 2019年1月—2020年1月长沙地区173 893份无偿献血标本共确认58例OBI样本,OBI流行率为0.033%;共发现7种血清学模式,抗HBc单独阳性最多,占38.98%,所有样本中抗HBc阳性率为89.83%;16例样本能检测出病毒载量,其中14例样本浓度小于100 IU/ml;抗HBs阳性组和阴性组间的病毒载量检出率无统计学差异;75.0%(12/16)样本扩增出S区序列,基因型均为B型,均发生突变,其中11例的HBsAg抗原决定簇及周边主要亲水区域(major hydrophilic region, MHR)发生氨基酸突变。结论 长沙地区无偿献血者中的OBI感染率在全国属于偏低水平;HBV基因型主要是B型,MHR区的氨基酸突变可能是造成OBI的原因,突变有本地特点。     

Evolutionary selection associated with the multi-function of overlapping genes in the hepatitis B virus

It is a challenge to understand the discrete roles of each point mutation in viral evolution, but overlapping genes provide an excellent entrance for the investigation of this complicated process. We obtained 132 sequences from the largest overlapping region in the HBV genome. Based on the genetic divergence between genotypes B and C, we distinguished a set of related footprint mutations that are believed to be responsible for historical selection events. Examining the mutations in the functional domains, we found that the virus has adopted a coherent strategy in its evolutionary process that can be summarized as follows: (1) the distribution of mutations was non-random throughout the overlapping region, and more mutations were preserved in the sequence when one of the genes was under relaxed selection; (2) the viral domains were subject to different selective pressures; for instance, the PreS1 domain underwent a strict selection, whereas the overlapped Spacer domain was relatively relaxed with obvious tolerance of non-synonymous mutations with a high dN/dS ratio; (3) different selective pressures on two codon sites ultimately determined that every mutation persevered at a proper position. Taken together, the functional constraints of protein domains are believed to be primarily responsible for the different selection patterns exhibited by the distribution of mutations and amino acid changes in the region where overlapping genes reside.     

拉米夫定早期疗效与乙型肝炎病毒基因型的关系

目的研究拉米夫定早期疗效与乙型肝炎病毒(HBV)基因型的关系。方法选取2001至2002年拉米夫定治疗12个月的595例慢性乙型肝炎患者作为研究对象,分别对HBV基因型和DNA(HBVDNA)、HBeAg／抗HBe及治疗12个月后的酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸(YMDD)变异进行检测,应用SPSS软件分析数据。结果595例慢性乙型肝炎患者中,A型8例(1．4％),B型53例(8．9％),C型360例(60．5％),BC混合型112例(18．8％),AC混合型14例(2．4％),AB混合型15例(2．5％),ABC混合型6例(1．0％),未分型者27例。拉米夫定治疗12个月后HBVDNA阴转率：B型87．2％,C型89．51％,BC型93．04％,三者比较,差异无统计学意义;HBeAg血清转换率：B型11．65％,C型20．64％,BC混合型18．57％,三者比较,差异无统计学意义;治疗12个月时HBVDNA出现反跳的病例中,69例发生了YMDD变异,其中C型46例(15．38％),B型9例(16．98％),BC混合型14例(13．86％),差异无统计学意义。结论拉米夫定早期疗效与HBV基因型、HBeAg血清转换率、HBVDNA水平及YMDD变异未见直接关系。     

乙肝基因分型与耐药位点突变的关系

目的:检测本地区乙肝病毒复制病人中基因型分布情况,探讨HBV基因型与抗病毒疗效关系。方法:应用乙肝病毒型特异性引物采用巢式PCR方法,对45例HBV-DNA阳性患者进行HBV基因型分析,用DNA测序方法检测耐药位点突变。结果:B型7例,占15.56%;C型34例,占75.56%;未分型3例,占6.67%;B+C混合型1例,占2.22%。未发现其他型别。C型病人人均病毒载量5.67±1.61(1oglncopies/m1)较B+C混合型5.54(只有1例)、B型(4.42±1.39)及未分型(2.65±0.57)高。C型病人耐药变异位点多,病人比例高。结论:浙江等地区HBV—DNA基因分型以C、B为主,而B基因型抗病毒治疗疗效最好,C型、混合型及其它型对抗病毒治疗药物疗效较差。